Anti-herpesviral agents and assays therefor

ABSTRACT

There is described an antiviral agent capable of disrupting the association of two viral structural proteins required for maturation, replication and infection of herpesviruses. The agents are based upon VP22 and disrupt the normal association of that protein with VP16 and/or gB. Suitable agents are peptides having the amino acid sequences TPRVAGFNKRVFCAAVGRLAAMHARMAAVQLW or ITTIRVTVCEGKNLLQRANE. The agents are suitable for combatting infection of herpesviruses and thus for the treatment of cod sores, genital herpes, chickenpox and shingles. An assay to test for agents able to disrupt VP22/V16 and/or VP22/gB association is also described.

This application is the U.S. national phase application of PCTInternational Application No. PCT/GB97/02036 filed Jul. 28, 1997.

The present invention relates to an anti-viral agent effective againstherpesviruses and to an assay for screening for other suitableanti-viral agents.

Herpesviruses are a large family of viruses which infect a wide range oforganisms. The term “Herpesvirus” is used herein to refer to any virusof the Herpes family, including viruses in the a group (e.g. HSV, PrV),the β group (eg HCMV) and in the γ group (eg EBV). Seven herpesvirusesare known to infect humans and there is evidence for an eighth humanherpesvirus. The most highly characterised human herpesvirus is herpessimplex type 1 (HSV-1) which is associated with causing lesions aroundthe mouth (cold sores). HSV-2, which is closely related to HSV-1, is aprimary cause of genital infections. A common feature of herpesvirusesis their ability to establish latent infections and recurrences of HSV-1and HSV-2 infections are common among infected individuals. For asizeable proportion of these individuals, recurrences are highlydebilitating and impact upon quality of life. In other situations, HSV-1and HSV-2 infection can be life-threatening. A third related virus,varicella zoster virus (VZV), is the causative agent of chickenpox inchildren and shingles in adults.

Herpesvirus virions consist of four morphologically distinct components,the core, capsid, tegument and envelope (reviewed in Rixon, 1993).

In virions made by HSV-1, the prototype a-herpesvirus, there are about29 viral polypeptides in the tegument and envelope (15 to 18polypeptides in the tegument and 11 glycoproteins in the envelope). Thusthese two regions of virus particles account for more than 30% of thegenes encoded by the virus genome. From studies on L-particles, whichare virus-related particles that lack a nucleocapsid and are made byHSV-1, it has been demonstrated that the tegument and envelope cancombine to assemble mature particles whose properties areindistinguishable from those of virions during the early events afterinfection (Szilágyi and Cunningham, 1991; McLauchlan et al., 1992; Rixonet al., 1992). The compositions of the tegument and envelope in virionsand L-particles are also very similar (Szilágy! and Cunningham, 1991;McLauchlan and Rixon, 1992), hence, interaction with the capsid is not aprimary determinant for incorporation into either of thesesub-structures of virions. It follows that interactions between thetegument and envelope components play a critical role in particleassembly and maturation.

Three of the most abundant structural proteins are glycoprotein B (gB),VP16 and VP22. gB is located in the envelope while VP16 and VP22 aretegument proteins.

VP16 is the product of the UL48 gene and is 490 amino acid residues inlength with an apparent molecular weight of 65 KDa on denaturingpolyacrylamide gels. This protein plays an essential role in bothactivation of transcription of immediate early (IE) genes and theassembly of the progeny virions (Weinheimer et al., 1992; reviewed inO'Hare, 1993). Hence, deletion of this gene abrogates virus growth and,to date, it is the only tegument protein known to be essential for virusgrowth. Mutagenesis of the UL48 gene demonstrated that distinct regionsof the VP16 protein are involved in transactivation and assembly (Ace etal., 1988). The sequences involved in transactivation can be separatedinto two domains. One domain, within the N-terminal portion of theprotein, is specific for protein interactions with cellulartranscription factors. Another domain is located within the C-terminaltail region of the polypeptide; this region is rich in acidic residues,however, apart from HSV-2, it is not conserved in homologues of VP16.

The function of the other major tegument protein, VP22, has not beenwell characterised. The protein is encoded by the UL49 gene (Elliott andMeredith, 1992) and the open reading frame (ORF) consists of 301 aminoacid residues. On denaturing polyacrylamide gels, the protein has anapparent molecular weight of approximately 38 KDa. In infected cells, itis extensively modified post-translationally by phosphorylation,poly(ADP)ribosylation and nucleotidylylation (Blaho et al., 1994).Immunofluorescence studies have shown that, in infected cells, VP22 islocated in the cytoplasm with high concentrations around the nuclearmembrane (Elliott and Meredith, 1992). It also associates with thenuclear matrix and therefore may have DNA-binding ability (Knopf andKaerner, 1980). Recent evidence has revealed that VP22 has the abilityto exit and re-enter cells although the mechanism which mediates thisproperty is unknown (Elliott and O'Hare, 1997). Within the tegument,VP22 is the most abundant structural protein and recent evidence hasshown that its abundance in the tegument can be further enhanced byaltering the amount of VP22 produced during infection (Leslie et al.,1996). we have evidence that mutations within this protein significantlyreduce virus growth (J. McLaughlan and Y. Sun, unpublished data). In arelated bovine herpesvirus, the removal of the gene that encodes theprotein homologous to VP22 severely impairs virus growth (Liang et al.,1995).

gB is the most abundant of the envelope components. It is encoded bygene UL27 and is the most highly conserved gene among those encodingherpesvirus glycoproteins. Along with three other glycoproteins (gD, gHand gL), it is essential for virus replication in tissue culture and isrequired for virus penetration and cell to cell spread. The unprocessedpolypeptide consists of 904 residues and, on denaturing polyacrylamidegels, the mature species has an apparent molecular weight of about 120KDa. The encoded polypeptide can be separated into four domains: acleavable signal sequence of 30 residues, an ectodomain (externaldomain) of 697 residues, a hydrophobic transmembrane domain of 68 aminoacids and an extensive endodomain (cytoplasmic region) of 109 aminoacids (Cai et al., 1988). The cytoplasmic domain is reported to have arole in cell-cell fusion and this is supported by the mapping of synmutations to this region (Bond et al., 1982; Gage et al., 1993). Thebiologically active form of gB is an oligomer. Two discontinuous sitesfor oligomer formation have been characterised, a non-essential regionin the N-terminal portion of the mature polypeptide and an essentialsite proximal to the membrane-spanning domain (Highlander et al., 1991;La Querre et al., 1996). Defective forms of gB, which retain the abilityto form hetero-oligomers, inhibit complementation of gB null mutants bythe wild-type gB molecule and thus exhibit negative transdominance (Caiet al., 1988). Among the mutants which display this property areC-terminally truncated forms which retain the transmembrane domain andthe regions required for oligomerisation but lack the cytoplasmic tail.

Following treatment of virus particles with a cross-linking reagent,four structured proteins, which were not present on the virus envelope,were co-precipitated with gB using a gB-specific polyclonal antiserum(Zhu and Courtney, 1994); this suggested that, in the virus particle, gBis in close proximity to these proteins. One of these proteins wasimmunologically characterised to be VP16, two were tentativelyidentified as VP11/12 (encoded by gene UL46) and VP13/14 (encoded bygene UL47) and the fourth was not classified but did have the sameapparent molecular weight as VP22. From the topography of gB, it isreasonable to speculate that the cytoplasmic domain of the protein mayinteract with tegument proteins underlying the envelope. Blocking anyinteraction of the C-terminal domain of gB with tegument proteins mayinhibit incorporation of the protein into virions, thus generating viruswith either no or reduced infectivity. This could be achieved throughbinding of a peptide or a peptide derivative to the intracellular domainof wild type gB.

Recent studies have shown that VP16 and VP22 also interact (Elliott etal., 1995). This interaction is detected in infected cells byimmunoprecipitation of the complex by a VP16-specific antibody.Interestingly, co-expression of VP16 and VP22 in transfected cells, inthe absence of other HSV proteins, leads to relocalisation of bothproteins to novel spherical structures termed tegument bodies.Experiments with baculovirus recombinants expressing these proteins haverevealed that indistinguishable structures are produced in insect cells(J. McLauchlan and F. J. Rixon, unpublished data). Thus, tegument bodiesare likely to result from the interaction between VP16 and VP22.

In addition to the formation of virus particles, tegument proteins alsohave a role during the initial stages of infection. Hence, inhibitingthe function of tegument proteins has the potential for disabling theinfectious process both during virus assembly and at some other stage ofinfection.

The action of VP16 requires intimate involvement with other proteins andthus the complex formed with VP22 could be crucial to either or both ofthe functions assigned to VP16. The region of VP16 which is involved inthis interaction is at the C-terminus of the protein and this is thedomain that has a role in activating the IE viral genes.

gB, VP16 and VP22 have been described previously in the literature.McGeoch et al. (1988) disclosed the whole nucleotide sequence and thepredicted amino acid sequences of HSV-1 strain 17 including genes UL27,UL48 and UL49 which encode gB, VP16 and VP22 respectively. All 3 genesare leftward orientated on the prototype orientation of the virusgenome.

The nucleotide sequence of HSV-1 strain 17, containing the full codingsequences of gB, VP16 and VP22, is available from publically accessibledatabases under Accession Number X14112.

The construction of clones of gB, VP16 and VP22 nucleotide codingsequences is well within the scope of abilities of the skilled man, andsuch coding sequences could be generated de-novo by DNA synthesis orderived from publically accessible clones by established PCR techniques.

The present example describes interactions which occur between gB andVP22 and between VP16 and VP22. Using truncated forms of these proteinswhich have been expressed in bacteria, the regions involved in theinteractions have been located to the C-terminal 107 residues of gB (theendodomain of the protein), a 109 residue region of VP22 encoded bynucleotides 105590 to 105919 of HSV-1 (hereafter termed the C-proximalregion of VP22) and the N-terminal 412 residues of VP16. Associationbetween VP22 and gB had not been established previously.

As is further described in the examples, synthetic peptides (A to J;Table 1) have been tested for their ability to interfere with theassociation between VP22 and VP16 or between VP22 and gB and suitableassays have been developed. We have found that peptides D and E preventassociation of VP22 and VP16 and also prevent association of VP22 andgB. A further peptide, peptide H, is capable of binding to VP16, butwhilst it does not prevent interaction with VP22, peptide H does inhibitVP22/gB association. One explanation of this observation is the presenceof two sites on VP22 where VP16 and gB may interact. A combination ofantiviral agents able to disrupt association at these two putative sitescould be advantageous.

According to the present invention there is provided an antiviral agentcapable of combatting maturation and/or replication of a herpesvirus bydisrupting association of VP22 with VP16 and/or gB.

A suitable agent would be the highly conserved oligopeptideTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLw (encoded from nucleotides 105728 to105823 on the HSV-1 genome corresponding to the gene UL49), or a portionor functional equivalent thereof. In particular, the oligopeptidesTPRVAGFNKRVFCAAVGRLA (peptide D) and CAAVGRLAAMHARMAAVQLW (peptide E)have been found to prevent association of VP22 with VP16 and/or VP22with gB.

A second suitable agent would be the oligopeptide ITTIRVTVCEGKNLLQRANE(encoded from nucleotides 105621 to 105680 on the HSV-1 genomecorresponding to the gene UL49), or a portion or functional equivalentthereof. This oligopeptide (peptide H) has been found to bind to VP16and to prevent association of VP22 with gB.

The portion of VP22 identified above has been predicted to comprise ahelix. It is possible that the secondary structure is of equal orgreater importance for the binding to VP16 and gB than the precisenucleotide or amino acid sequence. The present invention thusencompasses variants or mutations of the above VP22 domain which have nosubstantial effect on the binding function.

The anti-viral agent may be a peptide (for example, the peptidesindicated above) or a peptidomimetic compound which would be resistantto enzymic breakdown by peptidases. Peptidomimetic compounds of peptidesA-H (especially peptides D, E and H) form part of the invention.

The antiviral agent can preferably prevent either assembly of infectiousvirus particles or the activation of virus genes or the infectivity ofprogeny virus. The most widely used conventional anti-HSV compound, andmuch of the current development of other therapies, relies on theinterruption of DNA replication to block virus growth. Compounds whichare active at other stages of the virus growth cycle have the potentialto act in concert with, or independently from, conventional therapies.In addition, since homologues of the genes encoding gB, VP16 and VP22are present in other a-herpesviruses, anti-HSV compounds could beeffective against or further developed for treatment of other conditionssuch as chickenpox or shingles caused by VZV.

The anti-viral agent may be a peptide, either synthetic or derivedwholly or partially from a natural protein. Suitable anti-viralcompounds include peptides having an amino acid sequence derived fromVP22 (especially the C-proximal region of VP22) or a functionalequivalent of such a peptide. Peptidomimetic compounds therefor may besuitable anti-viral agents. The agent preferably binds to at least aportion of either gB or the VP16 C-terminus.

In a further aspect, the present invention provides an assay todetermine the ability of a test substance to interfere with theassociation of VP16 and VP22 or with the association of gB and VP22. Theassay comprises the following steps:

i) providing a first viral component;

ii) exposing said first viral component to a test substance followed bya second viral component, or exposing said first viral component to asecond viral component followed by a test substance;

iii) washing to remove any second viral component and/or test substancenot associated with the first viral component; and

iv) detecting the presence, and optionally determining the amount, ofsecond viral compound associated with said first viral component.

The first or second viral components may be localised on a surface, suchas a blotting membrane, or an assay plate for ELISA etc. Preferably thefirst viral component is immobilised in such a manner, although theinvention contemplates the possibility of the assay being carried out insolution.

The first viral component may be gB, VP16 or VP22. Where the first viralcomponent is either gB or VP16, the second viral component will be VP22.Where the first viral component is VP22, the second viral component willbe either VP16 or gB.

Detection of the presence and/or amount of second viral componentassociated with the first viral component may be conducted by anyconvenient means. Generally detection may be via an antibody (preferablymonoclonal), the presence of which can be established by exposure to asecond labelled antibody (again preferably monoclonal) in a typicalELISA-style assay, although direct labelling of the first antibody (oreven one of the viral components) is possible.

The invention also provides a method of combatting viral maturationand/or replication of a herpesvirus, the method comprising providing anagent capable of interfering with the interaction of gB and/or VP16 withVP22.

The invention also provides the use of an agent capable of interferingwith VP16/VP22 association or with gB/VP22 association for combattingherpesvirus infection, replication or maturation, and for themanufacture of a medicament for combatting herpesvirus infection,replication or maturation.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 (A) Relevant features of the pYS360 construct. The map shows thelocations of the T7 promoter and terminator sequences which controlexpression of VP22trunc. The order of the elements which-compriseVP22trunc is shown and Kan represents the position of the kanamycinresistance gene.

(B) The predicted sequence of VP22trunc. The regions of the polypeptidethat are not derived from VP22 but which contain the histidine andepitope tag motifs are underlined. The sequence is given in SEQ ID No 3.

FIG. 2 Molecular weight determination of VP22trunc by FPLC. 200pl ofVP22trunc at a concentration of 0.5 mg/ml was applied to a Superdex 7510/30 column. The column was run at a flow rate of lml/min. The point atwhich the sample was applied to the column is arrowed. Proteins weredetected by absorption at 280 nm. The molecular weight of VP22 wasdetermined by comparison with the relative mobilities of marker proteinsof known sizes. These were: lysozyme (14 KDa), trypsin (24 KDa),carbonic anhydrase (29 KDa), pepsin (35 KDa) and BSA (66 KDa).

FIG. 3 Quantitative analysis of purified GST-gB fusion protein. Proteinswere separated on a 12% polyacrylamide gel and then stained withCoomassie Brilliant blue. Samples were as follows: lane 1, 10 μg of BSA;lane 2, 5 μg of BSA; lane 3, 2.5 μg of BSA; lane 4, 1.25 μg of BSA; lane5, molecular weight markers; lane 6, 10 μl of purified GST-gB; lane 7, 5μl of purified GST-gB; lane 8, 10 μl of purified GST; lane 9, 5 μl ofpurified GST. The sizes of polypeptides (in KDa) are indicated.

FIG. 4 Co-elution of VP16 with VP22trunc from Ni-NTA resin. Partiallypurified extract containing VP16 was incubated in the absence of (lane14) or presence of 10 gg of VP22trunc (lanes 3 to 13). In lanes 3 to 12,an equal volume of the individual peptides at 2 mg/ml was added to theextract prior to VP22trunc. Peptides added to each reaction were asfollows: lane 3, peptide A; lane 4, peptide B; lane 5, peptide C; lane6, peptide D; lane 7, peptide E; lane 8, peptide F; lane 9, peptide G;lane 10, peptide H; lane 11, peptide I; lane 12, peptide J; lane 13, nopeptide. Lanes 3 to 14 show the polypeptides eluted from Ni-NTA resin.Other samples were as follows: lane 1, partially purified VP16 extract,lane 2, purified VP16; lane 15, purified VP22trunc. Samples wereelectrophoresed on a 12% polyacrylamide gel and the apparent molecularweights of VP16 (65 KDa) and VP22trunc (16 KDa) are shown.

FIG. 5 Far Western blot analysis of VP16 binding to VP22.

(A) Binding of VP116 to immobilised VP22trunc. Purified VP22trunc wasadded to partially purified VP16 extract and the sample was run on a 12%polyacrylamide gel. Proteins were then transferred to nitrocellulosemembrane and the blot was cut into strips, with each strip containing atleast 2 μg of VP22trunc. Strips were incubated with no protein (lane 1),2 μg of purified VP16 (lane 2) or 2 μg of purified VP16trunc (lane 3);bound VP16 was detected by antibody LP1 (1:1000 dilution). In lane 4,the membrane was incubated with the 9220 antibody (1:1000). The apparentmolecular weights of VP16 (65 KDa) and trimer (48 KDa), dimer (32 KDa)and monomer (16 KDa) forms of VP22trunc are shown.

(B) Binding of VP16 to truncated forms of VP22 expressed in bacteria.Samples were electrophoresed on a 12% polyacrylamide gel and thenproteins were transferred to nitro-cellulose membrane. Samples were asfollows: lane 1, uninduced extract of VP22/172-259; lanes 2 and 6VP22/159-301; lane 3, VP22/159-301mut; lanes 4 and 7, VP22trunc; lanes 5and 9 VP22/172-259; lane 8, VP22/159-259. Lanes 2 to 5 contain crudeextracts in which expression has been induced. Lanes 6 to 9 containproteins purified on Ni-NTA resin. The blot was incubated with VP16 (2mg/ml), followed by LP1 antibody (1:1000 dilution). The apparentmolecular weights (in KDa) of the truncated forms of VP22trunc areshown.

FIG. 6 ELISA of VP16 binding to VP22trunc. Microtitre wells were coatedwith a range of quantities of VP22trunc in duplicate (0 ng, 20 ng, 40ng, 80 ng, 160 ng and 320 ng). After blocking, VP16 was added at variousconcentrations and then detected with LPI antibody at a 1:1000 dilution.The legend for the concentrations of VP16 added is shown to the right ofthe graph. Data points were determined by calculating the average valueof duplicates. The data point obtained with the concentrations of VP22and VP16 which were used in subsequent ELISA tests is arrowed.

FIG. 7 Far Western analysis of the ability of peptides to block theinteraction between VP16 and immobilised VP22trunc. Purified VP22truncwas added to partially purified extract of VP16 and the sample run on a12% polyacrylamide gel. Proteins were then transferred to nitrocellulosemembrane and the blot was cut into strips, with each strip containingapproximately 1 g of VP22trunc. In (A), strips were pre-incubated with 1mg of each of the following peptides: lane 2, no peptide, lane 3,peptide C; lane 4, peptide D; lane 5, peptide E; lane 6, peptide F; lane7, peptides D and E; lane 8, peptides C and F. In (B) strips werepre-incubated with 1 mg of each of the following peptides: lane 2, nopeptide, lane 3, peptide C; lane 4, purified peptide D; lane 5, purifiedpeptide E; lane 6, peptide F. 24 g of pure VP16 was then added to strips2 to 8 in (A) and strips 2 to 6 in (B), followed by incubation with LP1antibody (1:1000 dilution). As a control, portions of the blot wereincubated with LP1 or the 9220 antibody at a dilution of 1:1000 (lane 1for LP1 in A and B; lane 9 in A and lane 7 in B for 9220). The apparentmolecular weights of VP16 (65 KDa) and the dimer (32 KDa) and monomer(16 KDa) forms of VP22trunc are shown.

FIG. 8 Blocking of the interaction between VP16 and full length VP22 bypure peptides D and E. A vUL49ep L-particle extract was run on a 10%polyacrylamide gel and the proteins transferred to a nitrocellulosemembrane. The blot was cut into strips, with each strip containing theequivalent of approximately 3×10⁹ L-particles. Strips were pre-incubatedwith 1 mg of each of the following peptides: lane 2, no peptide; lane 3,peptide C; lane 4, peptide D; lane 5, peptide E. 2 μg of pure VP16 wasthen added to each incubation and bound VP16 was detected by LP1antibody. Two strips were incubated with either LPl (lane 1) or 9220antibody (lane 6), each at a dilution of 1:1000. The apparent molecularweights of VP16 (65 KDa) and tagged VP22 (40 KDa) are shown.

FIG. 9 Inhibitory effect of peptides D and E on the VP22trunc-VP16interaction in ELISAs. Microtitre plates were coated with 160 ng ofVP22trunc and blocked with PBS/10% NCS. Before addition to the wells,five-fold dilutions of the peptides, ranging from 500 μg/ml to 1 g/ml,were incubated with VP16 (1.6 μg/ml). Bound VP16 was detected with LP1at a dilution of 1:1000. The legend for the peptides added is shown tothe right of the graph. Values are shown relative to those obtained inthe absence of the peptide.

FIG. 10 Binding of VP16 to peptides. Microtitre plates were coated with5-fold dilutions of peptides ranging from 500 μg/ml to 1 μg/ml andblocked with PBS/10% NCS. VP16 was then added to a final concentrationof 1.6 μg/ml and detected with LP1. The legend for the peptides added isshown to the right of the graph.

FIG. 11 Far Western blot analysis of GST-gB binding to purified HSV-1virions and L-particles. Virus particles (approximately 3×10⁹ particlesper sample) were electrophoresed on a 15% polyacrylamide gel and blottedon to Problott membrane. Portions of the membrane were incubated witheither purified GST-gB (lanes 1, 3 and 4) or GST (lane 2) at a finalconcentration of 1.2 μg/ml. Bound protein was detected with anti-GSTantibody. Samples were as follows: lanes 1 and 2, HSV-1 strain Fvirions; lane 3, vUL49ep L-particles; lane 4, vUL49A268-301 L-particles.The apparent molecular weights of proteins are indicated.

FIG. 12 Far Western blot analysis of the interaction between GST-gB andVP22trunc. Purified VP22trunc was electrophoresed on a 15%polyacrylamide gel. Proteins were transferred to PVDF membrane and theblot was cut into strips, with each strip containing approximately 1-2μg of VP22trunc.

(A) Binding of GST-gB to VP22trunc. Strips were incubated with eitherpurified GST-gB (lane 1) or GST (lane 2) at a final concentration of 1.2μg/ml and the bound protein was detected with anti-GST antibody. In lane3, the membrane was incubated with 9220 antibody.

(B) Inhibition of binding of GST-gB (final concentration 1.2 μg/ml)andeach of the following peptides at a final concentration of 250 μg/ml:lane 1, no peptide; lane 2, peptide A; lane 3, peptide B; lane 4,peptide C; lane 5, peptide D; lane 6, peptide E; lane 7, peptide F; lane8, peptide G; lane 9, peptide H; lane 10, peptide I; lane 11, peptide J.Bound GST-gB was detected with anti-GST antibody.

The apparent molecular weights of VP22trunc (16 KDa) and non-specificspecies detected by GST-gB (65 KDa and 25 KDa) are shown.

SEQ ID No 1 Nucleotide and predicted amino acid sequence of the UL49gene which encodes VP22 (McGeoch et al., 1988) SEQ ID No 2 Predictedamino acid sequence from SEQ ID No 1 SEQ ID No 3 Predicted sequence ofVp22trunc SEQ ID No 4 Peptide A (see Table 1) SEQ ID No 5 Peptide B (seeTable 1) SEQ ID No 6 Peptide C (see Table 1) SEQ ID No 7 Peptide D (seeTable 1) SEQ ID No 8 Peptide E (see Table 1) SEQ ID No 9 Peptide F (seeTable 1)  SEQ ID No 10 Peptide G (see Table 1)  SEQ ID No 11 Peptide H(see Table 1)  SEQ ID No 12 Peptide I (see Table 1)  SEQ ID No 13Peptide J (see Table 1)

The present invention will now be described by way of 24 example withreference to the accompanying figures and to the following examples.

EXAMPLES Methods

Maintenance of Cells and Growth of Viruses.

BHK C13 cells were maintained in Glasgow modified Eagle's mediumsupplemented with 10% tryptose phosphate broth and 10% newborn calfserum.

The virus strains used in this study were HSV-1 wild-type strains 17(Brown et al., 1973) and strain F (Ejercito et al., 1968), vUL49ep(Leslie et al., 1996) and vUL49Δ268-301 (Leslie, 1996). For growth ofvirus, BHK cells were infected at a multiplicity of infection (m.o.i.)of {fraction (1/300)} PFU per cell. Following infection at 31° C. for 4days, the virus was harvested and virions and L-particles were purifiedon 5-15% Ficoll gradients as described by Szilagyi and Cunningham(1991).

Plasmids.

(i) VP22 constructs. The parent plasmid for the constructs whichexpressed the truncated forms of VP22 was pET28a (Novagen). This plasmidcontains T7 RNA polymerase promoter and terminator sequences. Thesetranscription control regions flank sequences which encode an ATGinitiation codon followed by a translated region that encodes a stretchof 6 histidine residues. Downstream from the sequences are uniquerestriction enzyme sites (NdeI and NheI) which are used for cloningpurposes. pET28a also contains the LacI gene to repress expression undernon-inducing conditions and the kanamycin resistance gene for antibioticresistance.

Plasmid pYS360 (FIG. 1A) was constructed by inserting a 380 bp HincIIDNA fragment from another plasmid pUL49Δ268-301 (Leslie, 1996) into theNheI site of pET28a. This fragment consists of nucleotides 521 to 845 ofthe UL49 gene (SEQ ID No 1) with an oligonucleotide inserted at position845 that encodes epitope tag sequences derived from the humancytomegalovirus UL83 gene (McLauchlan et al., 1994). PlasmidpVP22/159-259 was made by cleaving pYS360 with BssHII (position 803 inthe UL49 sequence, SEQ ID No 1) and BamHI (a site which lies immediatelyupstream of the T7 terminator) and replacing the fragment with anoligonucleotide which specifies amino acid residues 254 to 259immediately followed by a translational stop codon. PlasmidpVP22/172-259 was constructed by cleaving pVP22/159-259 with MscI(position 561 in SEQ ID No 1) and NdeI, filling in the overhanging 5termini and ligation. Plasmids pVP22/159-301 and pVP22/159-301mut weremade by inserting 460 bp MscI/EagI DNA fragments from pUL49ep (Leslie etal., 1996) and pUL49insl94 (Leslie, 1996) respectively into pYS360 whichhad been cleaved with MscI and NotI. Insertion of these fragmentsextended the region of VP22 expressed to the end of the open readingframe (amino acid 301). In both constructs, the HCMV UL83 epitope tagwas present following the VP22 sequences. pVP22/159-301mut alsocontained an oligonucleotide which encoded 4 amino acid residues thatwere inserted at the codon specifying amino acid 194 (nucleotideposition 626, SEQ ID No 1; Leslie, 1996).

(ii) VP16 constructs. Plasmid pETVP16 was used to express full-lengthVP16 under the control of the T7 RNA polymerase promoter (Arnosti etal., 1993). The truncated form of VP16 was expressed from a plasmidtermed pETVP16trunc (a gift from Dr C. Preston). To constructpETVP16trunc, a partially self-complementary oligonucleotide (5′GATCTAGTGAGAGCTCACTA-3′), yielding four overhanging bases at each end,was inserted into the unique BamHI site in pMC1Δin15-17. This plasmidlacks the VP16 sequences between the linker insertion sites in pMC1in115and pMC1in17 (Ace et al., 1988) with the BamH1 site lying immediatelyafter the codon specifying residue 412. The VP16 sequences were thenintroduced into plasmid pET8c to give pETVP16trunc.

(iii) GST-gB construct. The parent plasmid used to express thecytoplasmic tail of gB was pGex2TNMCR (a gift from Dr R Everett;Meredith et al., 1994) which is a derivative of a commercially availableconstruct pGex2T (Pharmacia). To construct pGex2TN.gB a MaeII/MseI DNAfragment (encompassing residues 53404 to 53044 on the HSV-1 genome;McGeoch et al., 1988) from plasmid pGX135 (consists of the HSV-1 KpnI nfragment in vector pAT153) was inserted into the SmaI site ofpGex2TNMCR. Insertion of this fragment at the SmaI site linked residues798 to 904 of gB to the glutathione-S-transferase (GST) proteinexpressed by pGex2TNMCR.

Antibodies.

The mouse monoclonal antibody 9220 (DuPont Ltd, UK) recognises a 10amino acid epitope derived from the HCMV UL83 gene product, which wasused to tag VP22 sequences. For detection of VP16, the mouse monoclonalantibody LP1 (a gift from A. Minson; McLean et al., 1982) was used. TheGST-gB fusion protein was detected using the IgG fraction of rabbitantiserum raised against glutathione-S-transferase (Sigma). Unlessotherwise stated, all antibodies were used at dilutions of 1:1000.

Bacterial Strains.

The VP16 and VP22 proteins were made in E.coli strain BL21(DE3). Thebacterial strain used to produce GST-gB was E.coli strain DH5a.

Production and Purification of Truncated Forms of Histidine-Tagged VP22.

BL21(DE3) cells containing the relevant plasmid DNA were grown overnightin 10 ml of YT medium containing 50 μg/ml kanamycin. This culture wastransferred to 1 liter of YT medium and grown for 3 hours at 37° C. Toinduce protein expression, the culture was put on ice for 3-5 minutes,IPTG was added to a final concentration of 50 μM and the culture wasincubated overnight at 15° C. Cells were spun down at 4,000 g for 10minutes and the pellet was resuspended in 30 ml binding buffer (20 mMTris-HCl, pH 8.0, 500 mM NaCl, 5 mM imidazole). The bacterial suspensionwas sonicated and centrifuged at 23,500 g for 20 minutes. Thesupernatant (called crude extract) containing the induced protein wasretained for further purification.

Proteins containing the histidine tag were purified by binding to nickelnitrilotriacetic acid resin (Ni-NTA, Qiagen). Crude extract was added toresin which had been equilibrated with binding buffer and binding of theHis-tagged VP22 to the resin occurred for 40 min at 4° C. The resin wasspun down at 800 g for 5 minutes, washed four times (20 minutes perwash) in 50 ml binding buffer and transferred to a column. To elutehistidine-tagged proteins, resin was washed with solutions containingincreasing concentrations of imidazole which competitively removes thebound proteins. Solutions containing 60 mM imidazole, followed by 100 mMand 200 mM imidazole in 20 mM Tris-HCl, pH 8.0, 500 mM NaCl were used. 1ml aliquots were collected and the amount of protein was determined byO.D. measurement at 280 nm. Protein was dialysed against 20 mM Tris-HCl,pH 8.0, 250 mM NaCl and concentrated at 7000 g using Centricon 10microconcentrators (Amicon).

Production and Purification of VP16.

BL21(DE3) cells containing the relevant plasmid DNA were grown overnightin 10 ml or 100 ml YT medium containing 250 μg/ml ampicillin. Thesecultures were transferred to either 1 liter or 10 liters of YT mediumand grown at 37° C. until the O.D. measured 0.5. To induce proteinexpression, IPTG was added to a final concentration of 1 mM and theculture was incubated for 2 hours at 26° C.

For studies of the interaction of VP22trunc with VP16 in solution, VP16was partially purified from bacteria. Cells were spun down at 4,000 gfor 10 minutes and lysed by sonication in 50 mM Tris-HCl, pH 8.2, 100 mMNa₂SO₄, 1 mM DTT, 10% glycerol, 0.1% CHAPS, 1 mM EDTA and 1 mM PMSF. Thelysate was dialysed at 4° C. against 50 mM Tris-HCl, pH 8.2, 100 mMNa₂SO₄, 1 mM DTT, 10% glycerol, 0.1% CHAPS, 1 mM EDTA and then clarifiedby centrifugation at 17,500 g. Soluble VP16 protein was partiallypurified by ion-exchange FPLC on a Mono Q column (Pharmacia) using aNaCl gradient from 50 mM to 500 mM. Fractions containing VP16 wereidentified by Western blot analysis using LP1 antibody. Peak fractionswere dialysed against 20 mM Tris-HCl, pH8.0, 250 mM NaCl at 4° C. andused thereafter without further purification.

For preparation of pure VP16 and truncated VP16 (VP16trunc), bacteriawere grown, induced and lysed as described above; however, the extractswere partially purified by precipitation with 30% w/v ammonium sulphate.Precipitated protein was resuspended in 50 mM MES(2-[N-morpholino]ethane sulphonic acid), pH 6.5, 50 mM NaCl, 100 mMNa₂SO₄, 10% glycerol, 0.1% CHAPS and applied to a Mono S ion exchangecolumn (Pharmacia). Protein was eluted by increasing the concentrationof NaCl. Fractions containing VP16 were identified and dialysed asdescribed above. The purity of protein was assessed to be >95% based onCoomassie Brilliant blue staining of denaturing polyacrylamide gels.

Production and Purification of GST-gB.

DH5α cells containing pGex2TN.gB plasmid were grown overnight at 37° C.in YT broth containing 100 μg/ml ampicillin. 6 ml of overnight culturewas used to seed a 500 ml culture of YT broth and this was shaken at37°C. for 4 hours. To induce expression of the fusion protein, IPTG wasadded to 0.1 mM and incubation at 37° C. was continued for 1 hour.

To prepare a crude extract, bacteria were pelleted by centrifugation at5,000 g for 15 minutes and the pellet was resuspended in 12 ml PBSAcontaining 1 mM PMSF and lmM EDTA. The resuspension was frozen at −20°C., thawed and then sonicated. The sonicated suspension was incubated onice and Triton X-100 was added slowly to a final concentration of 1%over a period of 20 minutes. Insoluble material was removed bycentrifugation at 10,000 g for 10 minutes at 4° C. and the supernatant,which was termed crude extract, was stored at −20° C.

For purification of GST-gB, a column of glutathione-agarose (Sigma),swollen and equilibrated in PBSA, was prepared and washed with 10volumes of a solution of PBSA, 1% Triton X-100, 1 mM PMSF and 1 mM EDTA.Crude extract was passed through the column which was then washed with10 vols. of PBSA. Bound protein was eluted with 50 mM reducedglutathione in 400 mM Tris-HCl, pH 8.0 and peak fractions containingGST-gB were stored at −20° C. Prior to probing membranes in Far Westernstudies, fractions were pooled and dialysed against PBSA containing aprotease inhibitor cocktail (Boehringer).

Synthesis and Purification of Oligopeptides. Peptides were synthesisedby continuous flow Fmoc chemistry (Atherton and Sheppard, 1989; McLeanet al., 1991) and, where stated in the text, were purified bypreparative reverse-phase HPLC (Owsianka et al., 1993). The Mr values ofpeptides were determined by fast atom bombardment mass spectrometry(M-Scan) and corresponded to the predicted values. Peptides weredissolved in 20 mM Tris-HCl, pH 8.0, 250 mM NaCl and centrifuged at11,500 g for 1 minute prior to use. Precipitates were observed withpeptides D, E, and G and these peptides were classified as partiallyinsoluble (Table 1). Precipitates were removed by centrifugation beforeuse.

TABLE 1 Sequences and properties of the synthetic peptides derived fromthe VP22trunc sequence Peptide Sequence SEQ ID No Mol. Wt. (Da) Purity(%) So1ubility A GSHMARTAPTRSKTPAQGLA 4 2037 87.8 soluble BKTPAQGLARKLHFSTAPPNP 5 2130 81.3 soluble C FSTAPPNPDAPWTPRVAGFN 6 214185.7 soluble D TPRVAGFNKRVFCAAVGRLA 7 2132 55.1 partly soluble ECAAVGRLAAMHARMAAVQLW 8 2125 58.8 partly soluble F RMAAVQLWDMSRPRTDEDLN 92403 99.0 soluble G PRTDEDLNELLGITTIRVTV 10 2254 62.2 partly soluble HITTIRVTVCEGKNLLQRANE 11 2257 90.4 soluble I NLLQRANELVNPDVVQDVPD 12 224772.6 soluble J DVVQDVPDPERKTPRVTGG 13 2065 93.7 soluble

Co-purification of VP16 with VP22trunc on Ni-NTA Resin. PurifiedVP22trunc and a partially purified extract containing VP16, both in 20mM Tris-HCl, pH8.0, 250 mM NaCl, were mixed at 4° C. for 15 to 30minutes on a rotator. 50 μl of equilibrated Ni-NTA resin was added toeach mixture and incubation was continued at 40C for a further 15 to 30minutes. Resin was pelleted at 800 g for 1 minute and the supernatant(non-bound fraction) was removed. Resin was washed sequentially with 0.5ml of 20 mM Tris-HCl, pH 8.0, 250 mM NaCl, 5 mM imidazole (twice) and0.5 ml of 20 mM Tris-HCl, pH 8.0, 250 mM NaCl, 60 mM imidazole (fourtimes). Bound protein was eluted by addition of 50 μl of boiling mix(160 mM Tris-HCl, pH 6.7, 2% SDS, 700 mM β-mercaptoethanol, 10%glycerol, 0.002% bromophenol blue) followed by heating to 100° C. for 5minutes. For peptide studies, peptides were dissolved in 20 mM Tris-HCl,pH8.0, 250 mM NaCl at a concentration of 2 mg/ml. Incubation of peptideswith partially purified VP16 extracts was performed at ambienttemperature for 2 hours prior to the addition of VP22trunc. Samples wereelectrophoresed on denaturing polyacrylamide gels.

Polyacrylamide Gel Electrophoresis.

Proteins were separated on gels containing 10%, 12% or 15% acrylamidecross-linked with 2.5% (wt/wt) N,N′ methylene bis-acrylamide.Polymerisation was initiated by addition of 0.04% TEMED and 0.06% APS.Samples were heated to 100° C. for 5 min in boiling mix prior to loadingon the gel. Electrophoresis was performed for approximately 1 hour at120-150 V or overnight at 40 V using the buffer system of Laemmli(1970). Proteins were detected by staining with Coomassie Brilliant bluefor 20 minutes followed by destaining, or were transferred tonitrocellulose membrane for further analysis.

Western Blot Analysis.

Following electrophoresis proteins were electrotransferred at 4° C. tonitrocellulose membrane (Hybond ECL, Amersham) in blotting buffer (25 mMTris-HCl, pH 8.3, 192 mM glycine, 20% methanol) for 5-6 hours at 50 mA.The membrane was then blocked overnight in TBS (20 mM Tris-HCl, pH 7.5,500 mM NaCl) containing 3% gelatin. This was followed by incubation withthe appropriate antibody at a dilution of 1:1000 in TTBS (TBS containing0.05% Tween 20) containing 1% gelatin for 1.5-2 hours. The membrane waswashed extensively with TTBS and bound antibody was detected by goatanti-mouse antibody (Sigma) at a dilution of 1:1000 in TTBS, 1% gelatin.After incubation for 1 hour with the secondary antibody, the membranewas washed with TTBS and incubated with enhanced chemiluminescence (ECL)buffer (Amersham) for 2 minutes then exposed to XS-1 film (Kodak).

Far Western Blot Analysis.

Following electrophoresis, proteins were electrotransferred to eitherHybond or ProBlott (Applied Biosystems) membrane and the membrane wasincubated in renaturation buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl,10% glycerol, 1 mM DTT) at 4° C. for 5-6 hours.

For membranes probed with either VP16 or VP22trunc, blocking wasperformed overnight as described for Western blot analysis. Probing withepitope-tagged VP22 or VP16 was performed in 20 mM Tris-HCl, pH 8.0, 250mM NaCl for 1.5 hours at ambient temperature. Excess probe was removedby washing 4 times (10 minutes per wash) in 20 mM Tris-HCl, pH8.0, 250mM NaCl, followed by incubation with the appropriate antibody at adilution of 1:1000 in TTBS/1% gelatin for 1.5-2 hours. Bound antibodywas detected as described for Western blot analysis.

For membranes probed with GST-gB, blocking was performed overnight inPBSA containing 1% non-fat dried milk and 0.05% Tween 20 at 4° C.Membranes were then washed twice in renaturation buffer (10 min/wash)prior to incubation with fusion protein (final concentration 0.5 μg/ml)in renaturation buffer containing 1% BSA. Incubation was again performedovernight at 4° C. Following washing with renaturation buffer (fourtimes, 10 minutes/wash) and a brief rinse with blocking buffer, themembrane was incubated with anti-GST antibody in PBSA, 1% non-fat driedmilk, 0.05% Tween 20 and 1% BSA for 1 hour at ambient temperature. Afterfour washes with PBSA, 0.05% Tween 20 (10 minutes/wash), bound antibodywas detected with secondary antibody (goat anti-rabbit IgG, wholemolecule; Sigma) conjugated to Horse Radish Peroxidase (HRP) byincubating at room temperature for 45 minutes. Following four furtherwashes with PBSA, 0.05% Tween 20, the secondary antibody was visualisedby enhanced chemiluminescence.

Fast Protein Liquid Chromatography (FPLC).

The sizes of proteins were determined on a Superdex 75 10/30 column (bedvolume 24 ml; Pharmacia) which was equilibrated with 20 mM Tris-HCl, pH8.0, 500 mM NaCl, 5% glycerol, 1 mM DTT. Protein samples, containingapproximately 0.1-0.2 mg protein, were applied to the system in a volumeof 200 μl. The samples were passed through the column at a flow rate of1 ml/minute and protein was detected at 280 nm. Samples were collectedin 0.2 ml fractions for further analysis.

Enzyme-Linked Immunosorbent Assays (ELISA).

Dilutions of proteins in PBS were coated overnight onto flat bottomedmicro-titre plates (Dynatech) at 37° C. and then blocked with either 2%BSA or 10% new-born calf serum (NCS) in PBS for 1 hour at 37° C.Specific binding of a second protein to the plate-bound protein wasperformed for 1.5-2 hours at ambient temperature. a Excess secondaryprotein was removed by washing four times with PBS, 0.3% Tween 20. Boundprotein was detected by incubation for 1.5-2 hours with LP1 antibody inPBS, 1% gelatin or PBS, 2% NCS. The bound antibody was detected byincubation for 1 hour at ambient temperature with anti-mouse antibodyconjugated to HRP (1:500) and visualised with Smg/ml enzyme substrate2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS; Sigma) incitrate-phosphate buffer containing 6 μl hydrogen peroxide in a totalvolume of 20 ml. Optical densities were measured on a Titertek MultiscanPLUS instrument.

RESULTS

1. Purification and Characteristics of Proteins Expressed in Bacteria

(i) The Truncated Forms of VP22.

Previous results had shown that VP16 and VP22 interact in HSV-1-infectedcells (Elliott et al., 1995). This interaction was reproduced inbiochemical studies in which in vitro-translated VP22 was co-purified onglutathione-Sepharose beads using a glutathione-S-transferase-VP16fusion protein that had been expressed in bacteria. To furthercharacterise the region within VP22 to which VP16 bound, a truncatedform of VP22, termed VP22trunc, which contained residues 159-267 of theprotein was expressed in bacteria. For purification and detectionpurposes, this segment of protein was flanked at the N-terminus with astretch of 22 amino acids which contained 6 consecutive histidineresidues and at the C-terminus by 13 amino acids that constituted anepitope tag derived from the human cytomegalovirus (HCMV) pp65 protein(FIG. 1B: SEQ ID No 3); the histidine residues allowed purification ofthe protein on Ni-NTA resin and the epitope tag could be recognised by amonoclonal antibody termed 9220. This expression system yieldedapproximately 5-8 mg of protein per liter of bacterial culture. Fromanalysis of the proteins eluted from Ni-NTA resin at differentconcentrations of imidazole, effectively pure, soluble protein (>95% asdetermined by Coomassie Brilliant blue staining of polyacrylamide gels)was obtained by elution with buffer containing 100 mM imidazole. Theauthenticity of VP22trunc was determined by Western blot analysis usingantibody 9220 (FIG. 5A, Lane 4) and by mass spectrometry (data notshown). Analysis of the molecular weight of native VP22trunc by sizeexclusion chromatography showed that approximately 70% of the proteinmade in bacteria was 33 KDa with the remaining 30% having a highermolecular weight (FIG. 2). Thus, most of the VP22 has a molecular weightthat corresponds exactly to twice the predicted size of monomericprotein and it was concluded that this 33 KDa species was a dimer. Thehigher molecular weight material is considered to be a mixture ofoligomeric forms of VP22trunc.

In addition to VP22trunc, four other truncated forms of VP22 wereproduced in bacteria (Table 2). The constructs which permitted synthesisof these polypeptides are described in Methods. Each protein waspurified using identical expression and purification methods as forVP22trunc. The only changes in characteristics observed were withVP22/159-259 and vP22/172-259 which eluted more efficiently from Ni-NTAresin in buffer containing 200 mM imidazole, and yields of vP22/172-259were much lower, probably due to difficulties with solubility.

TABLE 2 Features of the truncated forms of VP22 expressed in bacteriaTag VP22 Residues Attached to Polypeptide Mol.Wt.(KDa) Expressed ProteinVP22trunc 16 159-267 HCMV^(a) + histidine VP22159-301 20 159-301HCMV^(a) + histidine VP22159-301mut 20.5 159-301 (4 HCMV^(a) + aminoacid histidine insertion at 194) VP22159-259 14 159-259 histidineVP22172-259 12 172-259 histidine ^(a)denotes the HCMV epitope tag

(ii) VP16.

Two forms of VP16 were produced for studies on interactions with VP22.The first of these was full-length VP16 which was prepared in bothpartially purified and fully purified states (see Methods). PurifiedVP16 was shown to be authentic by Western blot analysis with monoclonalantibody LP1 (Fig SA, lane 1). In partially purified extracts, VP16could be identified as a 65 KDa species on Coomassie Brilliantblue-stained polyacrylamide gels (FIG. 4, lane 2). The truncated form ofVP16, VP16trunc was produced from plasmid pETVP16trunc in which thesequences encoding residues 413 to 490 are not expressed. This VP16product was purified to homogeneity in the same way as full length VP16and was recognised by LPI antibody (data not shown).

(iii) GST-gB.

The C-terminal amino acids of gB represent a charged domain of proteinwhich is located internally in the virus particle and hence may interactwith tegument proteins underlying the virus envelope. To examinepossible interactions with tegument proteins, and for purification anddetection purposes, these residues were linked to the GST protein whichhas a size of 26 KDa. Thus, the predicted size of the fusion protein wasabout 37 KDa. Following purification on glutathione-agarose beads, twopolypeptides with apparent molecular weights of about 35 KDa and 28 KDawere detected (FIG. 3, lanes 6 and 7); the upper species approximates tothe predicted size for the GST-gB fusion protein while the lower bandhas an identical apparent molecular weight to GST protein (lanes 8 and9). To further verify that the 35 KDa species was the fusionpolypeptide, Western blot analysis showed that anti-GST antibodyrecognised this protein (data not shown). Furthermore, the nucleotidesequence of the region containing gB sequences in pGex2TN.gB wasdetermined. This revealed no nucleotide changes as compared to thepublished sequence and verified that the gB sequences were in the sameopen reading frame as those for the GST gene. Therefore, it wasconcluded that the 35 KDa and 28 KDa species were the GST-gB fusionproduct and the GST protein respectively. It was assumed that the latterproduct was generated by proteolytic cleavage of the fusion proteinbetween the GST and gB domains which may have occurred during synthesisin bacteria. This is a consistent feature found in systemsover-expressing GST fusion proteins. Both species were routinely foundin purified preparations of the fusion protein and the relativeproportions of each were 1:1. The concentration of GST-gB in thispreparation, which was used in the experiments presented in Section 4,was about 250 μg/ml based on comparison with standard amounts of BSAprotein (FIG. 3, lanes 1-4).

2 In Vitro Analysis of the Interaction Between VP16 and VP22trunc.

(i) Co-purification of VP16 and VP22trunc on Ni-NTA Resin

The ability of VP16 to interact with VP22trunc was examined by mixingpurified VP22trunc with a bacterial extract containing VP16 (FIG. 4,lane 1) followed by analysis of the polypeptides retained on Ni-NTAresin (FIG. 4). Results revealed that in the absence of VP22trunc,several polypeptides were eluted from the resin (FIG. 4, lane 14). Inthe presence of VP22trunc, a novel band of 65 KDa also co-eluted (FIG.4, lane 13); Western blot analysis showed that this polypeptidecorresponded to VP16 (data not shown). This ability to specificallyelute VP16 only in the presence of VP22trunc was reproducible overseveral experiments using various quantities of VP22trunc and crudebacterial extract containing VP16. From these data, it was concludedthat the co-elution of VP16 with VP22trunc from Ni-NTA resin resultedfrom the specific interaction between these proteins.

(ii) Detection of VP22trunc by VP16 using Far Western Analysis

Previous investigations made use of Far Western analysis to study theinteraction between VP16 and VP22 (Elliott et al., 1995). In thosestudies, various forms of VP16 were separated by electrophoresis,blotted onto nitrocellulose filters and renatured. The blot was thenprobed with in vitro translated radio-labelled VP22 to detect binding toVP16. To extend our studies, Far Western analysis was used to examinewhether this interaction could be studied with VP22trunc attached to theblot and VP16 used as a probe. Binding of VP16 to proteins on the blotcould then be detected using LPI antibody. In FIG. 5A, purifiedVP22trunc has been added to a bacterial extract containing VP16 and theproteins electrophoresed on a polyacrylamide gel followed by transfer tomembrane. Probing individual strips from the membrane separately withLP1 and 9220 antibodies reveals the positions of VP16 (lane 1) andVP22trunc respectively (lane 4). In addition to monomeric VP22trunc,antibody 9220 also recognises the dimer and trimer forms of the protein;the presence of dimers in particular was a consistent observation andthese are thought to arise through incomplete denaturation of the nativeprotein. Incubation of a portion of the blot with VP16 followed by LP1reveals that not only does the antibody detect a 65 KDa proteincorresponding to VP16 but also a band corresponding to monomericVP22trunc (lane 2). Therefore, these data show that VP16 can recogniseVP22trunc immobilised on membrane. In the converse experiment, VP22trunccan also bind to immobilised VP16 (data not shown). To confirm thebinding specificity of VP16 for immobilised VP22trunc, a portion of theblot was probed with the VP16trunc. No binding of VP16trunc to VP22trunccould be detected (lane 3). These data confirm that removal of theC-terminal residues of the VP16 significantly reduces the ability ofVP16 to bind to VP22 (Elliott et al., 1995) and demonstrate the specificnature of the interaction between the proteins using this form ofanalysis.

To further define the region of VP22 involved in VP16 binding, studieswere performed with additional forms of bacterially-expressed VP22, twoof which lacked the C-terminal epitope tag (Table 2). Both purified andcrude extracts of all the available forms of VP22 made in bacteria wereelectrophoresed on a polyacrylamide gel and transferred tonitrocellulose membrane along with an uninduced bacterial extract. Theblot was incubated with VP16 and bound protein was detected with LP1.VP16 was able to associate with all of the VP22 species (Fig SB, lanes 2to 9). Furthermore, a polypeptide which contained residues 159-301 andhad an insertion of 4 amino acids at position 194 was recognised by VP16(FIG. 5B, lane 3). The additional bands detected in crude extractscontaining the VP22 proteins were present also in the uninduced controlsample (compare lane 1 with lanes 2-5). These data indicate that anamino acid sequence responsible for specifically binding VP16 liesbetween residues 172 and 259 of VP22 and that the epitope tag is notinvolved in the interaction.

(iii) Binding of VP16 to VP22trunc by ELISA

To adopt a more quantitative approach, an ELISA for VP16 binding toVP22trunc was developed. Optimal conditions were determined by coatingwells with various quantities of VP22trunc followed by blocking with 2%BSA. Plates were then incubated with dilutions of VP16 and the boundVP16 detected with LP1. This showed specific detection of VP16 bindingat a range of concentrations of VP16 and VP22trunc (FIG. 6). Based onthese data and repeat experiments (data not shown), the concentrationsof VP16 and VP22trunc used in subsequent assays were 1.64 g/ml and 3.2μg/ml respectively (FIG. 6, arrow).

3 Disruption of the VP16/VP22 Interaction by Synthetic Peptides

To further examine the region within VP22 to which VP16 binds and todetermine whether interaction between the proteins could be interrupted,a series of ten peptides were synthesised based on the predictedsequence of the VP22trunc polypeptide between residues 18 and 144 (Fig1B; SEQ ID No 3); this region encompasses the VP22 and epitope tagsequences in VP22trunc. Each peptide was 20 amino acids in length withan overlap of 8 residues between adjacent peptides. Relevantcharacteristics of the is peptides synthesised are given in Table 1.

(i) Inhibition of the VP16/VP22trunc Interaction in Co-purificationStudies.

Results presented in Section 2(i) had shown that VP16 co-elutes fromNi-NTA resin in the presence of VP22trunc and it was concluded that thisindicated interaction between these polypeptides. The ability ofsynthetic peptides to inhibit this interaction was analysed by mixingthem individually with the partially purified VP16 extract prior toaddition of VP22trunc. From the intensity of the 65 KDa species in thecrude extract (FIG. 4, lane 2), it was estimated that the concentrationof VP16 was approximately 0.5 nM. To ensure that experiments wereperformed in an excess of peptide, peptides were prepared at aconcentration of 2 mg/ml (about 1 mM) and equal volumes of peptide andextract were mixed. This gave a relative molar ratio of VP16 to peptideof 1:300. However, it should be noted that certain peptides were notcompletely soluble (Table 1) and in those cases, the ratio would bereduced. Analysis of the proteins which co-elute with VP22trunc in thepresence of each of these peptides is shown in FIG. 4. This revealedthat VP16 failed to co-elute with VP22trunc following incubation withpeptide E (lane 7) and in reduced amounts in the presence of peptide D(lane 6). By contrast, no quantitative differences in the amount of VP16which co-elutes were observed in the presence of the other peptides whencompared to the control sample (compare lanes 3 to 5 and 8 to 12 withlane 13). This suggested that peptides D and E could inhibit bindingbetween VP16 and VP22trunc. However, these peptide preparations were nothomogeneous (Table 1) and may contain impurities which non-specificallyinhibit the interaction. To eliminate this possibility, peptides D and Ewere purified to homogeneity by reverse phase HPLC and further analysisshowed that their inhibitory capabilities were retained (data notshown).

(ii) Inhibition of the VP16/VP22trun=Interaction in Far WesternAnalysis.

Based on the studies with soluble VP16 and VP22trunc presented above,only a restricted number of peptides were examined by Far Westernanalysis to identify those that may prevent binding of VP16. Thus,VP22trunc was added to a crude extract of bacterially expressed VP16 andthe proteins were electrophoresed on a polyacrylamide gel followed bytransfer to a membrane. As shown in FIG. 7A, incubation of a portion ofthe blot with VP16 followed by LP1 antibody identified two bands, one ofwhich corresponds to VP16.(compare lane 2 with lane 1) while the secondis the monomeric form of VP22trunc (compare lane 2 with lane 4). Beforethe addition of VP16 the blot strips in lanes 3 to 8 were incubated withcrude preparations of peptides C, D, E, or E′ as well as mixtures ofeither 0 and E or C and F. When the blot strips were incubated withpeptides C and F either singly or in combination, VP16 bindincg toVP22trunc was not prevented (FIG. 7A, lanes 3, 6 and 8). By contrast,incubation with peptides D and E either separately or as a mixtureresulted in loss of recognition of VP22trunc (FIG. 7A, lanes 4, 5 and7). In support of the co-purification studies in 2(i), these datasuggest that peptides D and E can block binding of VP16 to immobilisedVP22trunc. From Table 1 aLnd as described in above, impurities inpeptides D an(i E could account for their inhibitory effects. Therefore,the experiment as shown in FIG. 7A was repeated with purifiedpreparations of peptides D and E. Again, these peptides were able toblock binding of VP16 to VP22trunc (FIG. 7B, lanes 4 and 5) whilepeptides C and F had no effect (FIG. 7B, lanes 3 and 6). Theoligopeptide CAAVGRLA, comprising the overlap region between peptides Dand E, may be particularly important in the VP16 binding function, andthis oligopeptide, along with functional equivalents and substitutionsthereof, forms a further aspect of the invention.

The results presented thus far have established that peptides D and Eblock the interaction between VP16 and VP22trunc. The inhibitory effectof the peptides was further tested to examine whether they were capableof blocking the interaction of VP16 with full-length VP22. Hence, anextract from vUL49ep light particles containing full length VP22 waselectrophoresed on a polyacrylamide gel and the proteins w7ere thentransferred to membrane. vUL49ep is a recombinant HSV-1 virus whichexpresses two forms of VP22; the first is the endogenous form which isunmodified and the second is an epitope-tagged version which isexpressed under the control of the HCMV immediate early promoter (Leslieet al., 1996). Previous studies have indicated that the epitope-taggedversion of VP22 is present in high amounts in vUL49ep virus particles(Leslie et al., 1996). Incubation of blot strips with antibodies LP1 and9220 shows the positions of both VP16 and full-length VP22 respectivelyon the blot (FIG. 8, lanes 1 and 6). Probing with VP16 followed bit LP1shows that the antibody detects, in addition to VP16, a band of 40 KDawhich represents full-length tagged VP22 (FIG. 8, lane 2). Thisindicates that the residues containing the histidine tag present at theN-terminus of VP22trunc do not contribute to VP16 binding. Priorincubation with either peptide D or E completely blocks binding of VP16to VP22 (FIG. 8, lanes 4 and 5). However, peptide C does not hinder theinteraction (FIG. 8, lane 3). Thus, peptides D and E are sufficient tocompletely inhibit the recognition of both VP22trunc and full-lengthVP22 by VP16.

(iii) Inhibition of the VP16/VP22trunc Interaction in ELISAS.

The studies presented above have provided strong evidence in support ofpeptide inhibition of the interaction between VP16 and VP22. However,quantitative analysis of the ability of peptides to block anyinteraction is difficult to perform by the above methods. Therefore,using the ELISA system described in Results Section 2(iii) with theexception that PBS/10% NCS was utilised as a blocking agent, theinhibitory effects of peptides D and E were examined. This modificationarose due to nonspecific binding of the peptides to wells when BSA wasused for blocking (data not shown). Results showed that the addition ofthe pure preparations of either peptide D or E at concentrations of both100 and 500 μg/ml could inhibit binding of VP16 to VP22trunc (FIG. 9).However, neither peptides B, C nor F had any effect on interactionbetween the proteins (FIG. 9). 50% inhibition of VP16 binding wasobserved at concentration, of 212.5 μg/ml for peptide D and 85.7 μg/mlfor peptlde E. These correspond to molarities of 99.7 μM anti 44.1 μMrespectively for these peptides. It should be noted however, that theseconcentrations represent maximum molarities, since the peptides were not100% soluble even following purification.

(iv) Direct Binding of Peptides D anii E to VP16.

To examine whether VP16 could bind directly to the peptides, wells werecoated with each peptide (A-J) at a range of concentrations from 1 g/mlto 500 μg/ml and the plate was then blocked with PBS/13% NCS. Incubationwith VP16 showed that binding did not occur with peptides A, B, C, F, G,I and J (FIG. 10). However, binding was found with higher concentrationsof peptides D and E (FIG. 10). In addition, there was evidence also forVP16 binding to peptide H; the nature of this interaction was notfurther examined. Nonetheless, the data for peptides D and E implicatedirect binding of these peptides to VP16 as the mechanism for inhibitingits interaction with VP22. Peptide H also was active in experimentswhich prevented gB binding to VP22trunc [Section 4 (iii)] and thus alsoforms an aspect of the invention.

4 In vitro Binding of gB to VP22 and Disruption of Binding by SyntheticPeptides.

(i) Structural Proteins Recognised by GST-gB using Far Western BlotAnalysis.

In a previous study with cross-linking reagents, gB was found to be inclose proximity to four structural proteins in virions (Zhu andCourtney, 1994). Three of these proteins were proposed to be thetegument components, VP11/12, VP13/14 and VP16 although VP16 was theonly species positively identified by reactivity with a specificantibody. The fourth polypeptide had a similar molecular weight to VP22but was not considered to be a tegument protein. Since the tegumentunderlies the envelope, it is reasonable to conclude that theendodomains of glycoproteins will contact the tegument. To analysewhether the C-terminal residues of gB, which constitute the endodomainof the polypeptide, could interact with any structural components, FarWestern blot analysis was performed using purified HSV-1 virions withGST-gB as a probe. FIG. 11, lane 1 shows that a series of bands wereidentified following detection of bound GST-gB with anti-GST antibody.The major species had apparent molecular weights of 120 KDa, 90 KDa, 82KDa, 65 KDa and 38 KDa; similar data have also been obtained withL-particles (lane 3). Control experiments revealed that the 65 KDa bandis a non-specific species which was also detected using GST protein as aprobe (lane 2). Further data (not shown) have shown that the 90 KDa and82 KDa bands are VP11/12 (encoded by UL46) and VP13/14 (encoded by UL47)respectively. This agrees with the results obtained in the cross-linkingstudies performed by Zhu and Courtney (1994) although there is noevidence here that GST-gB associates with VP16. Presently, the 120 KDaband has not been characterised. In agreement with the data presented byZhu and Courtney (1994), the 38 KDa species has an identical molecularweight to VP22. To further characterise this species, GST-gB was used toprobe L-particles made by vUL49ep which contains epitope-tagged VP22and, moreover, the tagged VP22 is present in greater quantities whencompared with virus particles made by wild-type virus. This showed thatGST-gB bound to the same species as identified with wild-type virionsand, in addition, bound to tagged vP22 which has a slightly highermolecular weight than the natural protein (FIG. 11, lane 3). Anotherexperiment using L-particles made by a virus recombinant, vUL49Δ268-301,in which the C-terminal 34 residues have been removed from the taggedcopy of the UL49 gene, showed that GST-gB bound to this truncated formof VP22 (FIG. 11, lane 4). This provides direct evidence that theC-terminal region of gB interacts with VP22, and moreover that theC-terminal 34 residues of VP22 are not required for binding to gB.

(ii) Binding of gB to VP22trunc.

The above analysis demonstrated that gB binds specifically to VP22 andto a C-terminally truncated form of the protein. The ability of gB toassociate with the bacterially-expressed form of VP22, VP22trunc, wasthen studied. As shown in FIG. 12A, lane 1, three bands of 65 KDa, 25KDa and 16 KDa were detected following incubation with GST-gB andanti-GST antibody. Of these three species, the 65 KDa and 25 KDa bandsalso were evident in the control using GST protein and anti-GST antibody(FIG. 12A, lane 2). However, the 16 KDa protein was not observed andanalysis with 9220 MAb indicated that this polypeptide was VP22trunc(FIG. 12A, lane 3). Hence the region of VP22 consisting of amino acids159-267 not only binds VP16 but also interacts with the C-terminalresidues of gB.

(iii) Inhibition of gB/VP22trunc Binding by Synthetic Peptides.

Similar to the studies performed with inhibition of binding of VP16 toVP22, peptides A to J, which span residues 18 to 144 of VP22trunc, wereused to examine whether gB binding could also be prevented (FIG. 12B).Co-incubation of individual peptides with GST-gB showed that peptides D,E and H completely inhibited binding of gB (lanes 5, 6 and 9; theseinhibitory effects were reproducible in other experiments. The datapresented in Section 3(i-iii) also indicate that peptides D and Einhibit the interaction between VP16 and VP22 while there is evidencethat peptide H can bind VP16 [Section 3(iv)]. This suggests that thesepeptides have the ability, to interact not only with VP16, but also withthe C-terminal domain of gB.

Modifications and improvements can be incorporated without departingfrom the scope of the invention.

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14 1 950 DNA HERPESVIRUS TYPE 1 1 ggcctaattg tccgcgcatc cgaccctagcgtgttcgtgg aaccatgacc tctcgccgct 60 ccgtgaagtg cggtccgcgg gaggttccgcgcgatgagta cgaggatctg tactacaccc 120 cgtcttcagg tatggcgagt cccgatagtccgcctgacac ctcccgccgt ggcgccctac 180 agacacgctc gcgccagagg ggcgaggtccgtttcgtcca gtacgacgag tcggattatg 240 ccctctacgg gggctcgtca tccgaagacgacgaacaccc ggaggtcccc cggacgcggc 300 gtcccgtttc cggggcggtt ttgtccggcccggggcctgc gcgggcgcct ccgccacccg 360 ctgggtccgg aggggccgga cgcacacccaccaccgcccc ccgggccccc cgaacccagc 420 gggtggcgac taaggccccc gcggccccggcggcggagac cacccgcggc aggaaatcgg 480 cccagccaga atccgccgca ctcccagacgcccccgcgtc gacggcgcca acccgatcca 540 agacacccgc gcaggggctg gccagaaagctgcactttag caccgccccc ccaaaccccg 600 acgcgccatg gaccccccgg gtggccggctttaacaagcg cgtcttctgc gccgcggtcg 660 ggcgcctggc ggccatgcat gcccggatggcggcggtcca gctctgggac atgtcgcgtc 720 cgcgcacaga cgaagcactc aacgaactccttggcatcac caccatccgc gtgacggtct 780 gcgagggcaa aaacctgctt cagcgcgccaacgagttggt gaatccagac gtggtgcagg 840 acgtcgacgc ggccacggcg actcgagggcgttctgcggc gtcgcgcccc accgagcgac 900 ctcgagcccc agcccgctcc gcttctcgccccagacggcc cgtcgagtga 950 2 301 PRT HERPESVIRUS TYPE 1 2 Met Thr Ser ArgArg Ser Val Lys Cys Gly Pro Arg Glu Val Pro Arg 1 5 10 15 Asp Glu TyrGlu Asp Leu Tyr Tyr Thr Pro Ser Ser Gly Met Ala Ser 20 25 30 Pro Asp SerPro Pro Asp Thr Ser Arg Arg Gly Ala Leu Gln Thr Arg 35 40 45 Ser Arg GlnArg Gly Glu Val Arg Phe Val Gln Tyr Asp Glu Ser Asp 50 55 60 Tyr Ala LeuTyr Gly Gly Ser Ser Ser Glu Asp Asp Glu His Pro Glu 65 70 75 80 Val ProArg Thr Arg Arg Pro Val Ser Gly Ala Val Leu Ser Gly Pro 85 90 95 Gly ProAla Arg Ala Pro Pro Pro Pro Ala Gly Ser Gly Gly Ala Gly 100 105 110 ArgThr Pro Thr Thr Ala Pro Arg Ala Pro Arg Thr Gln Arg Val Ala 115 120 125Thr Lys Ala Pro Ala Ala Pro Ala Ala Glu Thr Thr Arg Gly Arg Lys 130 135140 Ser Ala Gln Pro Glu Ser Ala Ala Leu Pro Asp Ala Pro Ala Ser Thr 145150 155 160 Ala Pro Thr Arg Ser Lys Thr Pro Ala Gln Gly Leu Ala Arg LysLeu 165 170 175 His Phe Ser Thr Ala Pro Pro Asn Pro Asp Ala Pro Trp ThrPro Arg 180 185 190 Val Ala Gly Phe Asn Lys Arg Val Phe Cys Ala Ala ValGly Arg Leu 195 200 205 Ala Ala Met His Ala Arg Met Ala Ala Val Gln LeuTrp Asp Met Ser 210 215 220 Arg Pro Arg Thr Asp Glu Ala Leu Asn Glu LeuLeu Gly Ile Thr Thr 225 230 235 240 Ile Arg Val Thr Val Cys Glu Gly LysAsn Leu Leu Gln Arg Ala Asn 245 250 255 Glu Leu Val Asn Pro Asp Val ValGln Asp Val Asp Ala Ala Thr Ala 260 265 270 Thr Arg Gly Arg Ser Ala AlaSer Arg Pro Thr Glu Arg Pro Arg Ala 275 280 285 Pro Ala Arg Ser Ala SerArg Pro Arg Arg Pro Val Glu 290 295 300 3 144 PRT Artificial SequenceSYNTHETIC PEPTIDES DERIVED FROM THE VP22TRUNC SEQUENCE 3 Met Gly Ser SerHis His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly SerHis Met Ala Ser Thr Ala Pro Thr Arg Ser Lys Thr Pro 20 25 30 Ala Gln GlyLeu Ala Arg Lys Leu His Phe Ser Thr Ala Pro Pro Asn 35 40 45 Pro Asp AlaPro Trp Thr Pro Arg Val Ala Gly Phe Asn Lys Arg Val 50 55 60 Phe Cys AlaAla Val Gly Arg Leu Ala Ala Met His Ala Arg Met Ala 65 70 75 80 Ala ValGln Leu Trp Asp Met Ser Arg Pro Arg Thr Asp Glu Asp Leu 85 90 95 Asn GluLeu Leu Gly Ile Thr Thr Ile Arg Val Thr Val Cys Glu Gly 100 105 110 LysAsn Leu Leu Gln Arg Ala Asn Glu Leu Val Asn Pro Asp Val Val 115 120 125Gln Asp Val Pro Asp Pro Glu Arg Lys Thr Pro Arg Val Thr Gly Gly 130 135140 4 20 PRT Artificial Sequence SYNTHETIC PEPTIDES DERIVED FROM THEVP22TRUNC SEQUENCE 4 Gly Ser His Met Ala Arg Thr Ala Pro Thr Arg Ser LysThr Pro Ala 1 5 10 15 Gln Gly Leu Ala 20 5 20 PRT Artificial SequenceSYNTHETIC PEPTIDES DERIVED FROM THE VP22TRUNC SEQUENCE 5 Lys Thr Pro AlaGln Gly Leu Ala Arg Lys Leu His Phe Ser Thr Ala 1 5 10 15 Pro Pro AsnPro 20 6 20 PRT Artificial Sequence SYNTHETIC PEPTIDES DERIVED FROM THEVP22TRUNC SEQUENCE 6 Phe Ser Thr Ala Pro Pro Asn Pro Asp Ala Pro Trp ThrPro Arg Val 1 5 10 15 Ala Gly Phe Asn 20 7 20 PRT Artificial SequenceSYNTHETIC PEPTIDES DERIVED FROM THE VP22TRUNC SEQUENCE 7 Thr Pro Arg ValAla Gly Phe Asn Lys Arg Val Phe Cys Ala Ala Val 1 5 10 15 Gly Arg LeuAla 20 8 20 PRT Artificial Sequence SYNTHETIC PEPTIDES DERIVED FROM THEVP22TRUNC SEQUENCE 8 Cys Ala Ala Val Gly Arg Leu Ala Ala Met His Ala ArgMet Ala Ala 1 5 10 15 Val Gln Leu Trp 20 9 20 PRT Artificial SequenceSYNTHETIC PEPTIDES DERIVED FROM THE VP22TRUNC SEQUENCE 9 Arg Met Ala AlaVal Gln Leu Trp Asp Met Ser Arg Pro Arg Thr Asp 1 5 10 15 Glu Asp LeuAsn 20 10 20 PRT Artificial Sequence SYNTHETIC PEPTIDES DERIVED FROM THEVP22TRUNC SEQUENCE 10 Pro Arg Thr Asp Glu Asp Leu Asn Glu Leu Leu GlyIle Thr Thr Ile 1 5 10 15 Arg Val Thr Val 20 11 20 PRT ArtificialSequence SYNTHETIC PEPTIDES DERIVED FROM THE VP22TRUNC SEQUENCE 11 IleThr Thr Ile Arg Val Thr Val Cys Glu Gly Lys Asn Leu Leu Gln 1 5 10 15Arg Ala Asn Glu 20 12 20 PRT Artificial Sequence SYNTHETIC PEPTIDESDERIVED FROM THE VP22TRUNC SEQUENCE 12 Asn Leu Leu Gln Arg Ala Asn GluLeu Val Asn Pro Asp Val Val Gln 1 5 10 15 Asp Val Pro Asp 20 13 19 PRTArtificial Sequence SYNTHETIC PEPTIDES DERIVED FROM THE VP22TRUNCSEQUENCE 13 Asp Val Val Gln Asp Val Pro Asp Pro Glu Arg Lys Thr Pro ArgVal 1 5 10 15 Thr Gly Gly 14 32 PRT Artificial Sequence SYNTHETICPEPTIDES DERIVED FROM THE VP22TRUNC SEQUENCE 14 Thr Pro Arg Val Ala GlyPhe Asn Lys Arg Val Phe Cys Ala Ala Val 1 5 10 15 Gly Arg Leu Ala AlaMet His Ala Arg Met Ala Ala Val Gln Leu Trp 20 25 30

What is claimed is:
 1. An antiviral agent which prevents maturationand/or replication of an alphaherpesvirus by inhibiting association ofVP22 with VP16 and/or with gB, wherein said antiviral agent mimics aportion of HSV VP22, said portion being located at amino acids 159-301of HSV VP22.
 2. An antiviral agent as claimed in claim 1 which is apeptide having an amino acid sequence corresponding to the sequence ofthe C-proximal portion of VP22, wherein said C-proximal portion of VP22is the 109 amino acid portion encoded by nucleotides 105590 to 105919 ofHSV-1.
 3. An antiviral agent as claimed in claim 1 which binds to atleast a portion of the C terminus of gB and/or VP16.
 4. An antiviralagent as claimed in claim 1, wherein said agent is a peptide having asequence: a) TPRVAGFNKRVFCAAVGRLAAMHARMAAVQLW (SEQ ID No 14); or b)ITTIRVTVCEGKNLLQRANE (SEQ ID No 11); or c) a portion thereof.
 5. Anantiviral agent as claimed in claim 1 which is a synthetic peptide. 6.An antiviral agent as claimed in claim 1 which inhibits association ofVP22 with VP16.
 7. An antiviral agent as claimed in claim 1 whichinhibits association of VP22 with gB.
 8. A combination of an antiviralagent as claimed in claim 7 together with an antiviral agent whichprevents maturation and/or replication of a herpesvirus by inhibitingassociation of VP22 with gB.
 9. A combination of antiviral agents asclaimed in claim 8 comprising: a) the peptide TPRVAGFNKRVFCAAVGRLA (SEQID No 7) or a portion thereof; or b) the peptide CAAVGRLAAMHARMAAVQLW(SEQ ID No 8) or a portion thereof, together with the peptideITTIRVTVCEGKNLLQRANE (SEQ ID No 11) or a portion thereof.
 10. An assayto determine the ability of a test substance to interfere with theassociation of VP22 with VP16 or of VP22 with gB in an alphaherpesvirus,said assay comprising: i) providing a first viral component consistingof VP22; ii) providing a second viral component selected from the groupconsisting of gB and VP16; iii) exposing one of said first and secondviral components to a test substance followed by the other of said firstand second viral components, or exposing said first viral component tosaid second viral component followed by a test substance; iv) washing toremove any unassociated first or second viral component and/or testsubstance; and v) detecting the presence, and optionally determining theamount, associated first and second viral components.
 11. An assay asclaimed in claim 10 wherein one of said first and second viralcomponents is localised on a surface.
 12. An assay as claimed in claim10 wherein an antibody is used to detect the presence of second viralcomponent associated with said first viral component.
 13. A method ofpreventing maturation of an alphaherpesvirus, the method comprisingproviding an agent which inhibits the interaction of gB and/or VP16 withVP22, adding said agent to said replicating alphaherpesvirus insufficient quantity to cause said inhibition and monitoring the effecton viral replication and thus determining the presence or extent of saidinhibition.